Caffeic Acid Phenethyl Ester (CAPE) Inhibits Cross-Kingdom Biofilm Formation of Streptococcus mutans and Candida albicans

ABSTRACT Streptococcus mutans and Candida albicans exhibit strong cariogenicity through cross-kingdom biofilm formation during the pathogenesis of dental caries. Caffeic acid phenethyl ester (CAPE), a natural compound, has potential antimicrobial effects on each species individually, but there are no reports of its effect on this dual-species biofilm. This study aimed to explore the effects of CAPE on cariogenic biofilm formation by S. mutans and C. albicans and the related mechanisms. The effect of CAPE on planktonic cell growth was investigated, and crystal violet staining, the anthrone-sulfuric acid assay and confocal laser scanning microscopy were used to evaluate biofilm formation. The structures of the formed biofilms were observed using scanning electron microscopy. To explain the antimicrobial effect of CAPE, quantitative real-time PCR (qRT–PCR) was applied to monitor the relative expression levels of cariogenic genes. Finally, the biocompatibility of CAPE in human oral keratinocytes (HOKs) was evaluated using the CCK-8 assay. The results showed that CAPE suppressed the growth, biofilm formation and extracellular polysaccharides (EPS) synthesis of C. albicans and S. mutans in the coculture system of the two species. The expression of the gtf gene was also suppressed by CAPE. The efficacy of CAPE was concentration dependent, and the compound exhibited acceptable biocompatibility. Our research lays the foundation for further study of the application of the natural compound CAPE as a potential antimicrobial agent to control dental caries-associated cross-kingdom biofilms. IMPORTANCE Severe dental caries is a multimicrobial infectious disease that is strongly induced by the cross-kingdom biofilm formed by S. mutans and C. albicans. This study aimed to investigate the potential of caffeic acid phenethyl ester (CAPE) as a natural product in the prevention of severe caries. This study clarified the inhibitory effect of CAPE on cariogenic biofilm formation and the control of cariogenic genes. It deepens our understanding of the synergistic cariogenic effect of S. mutans and C. albicans and provides a new perspective for the prevention and control of dental caries with CAPE. These findings may contribute to the development of CAPE as a promising antimicrobial agent targeting this caries-related cross-kingdom biofilm.

much toxicity in the assay might translate to toxicity in vivo. Please either remove claims of "good biocompatibility" or add references explaining what "good biocompatibility" is and how the results here compare to it. 8. Line 218: please remove "promising", I think that's a bit overboard at this stage. 9. Lines 251-257: see comment #6 above 10. Line 265: do you mean "plants" instead of "implants"? 11. Line 277: see comment #7 above. 12. Lines 371-372: Since I don't believe that YPD and BHI are selective, how do you differentiate between colonies of S. mutans and C. albicans by plating on solid media from the co-culture? This is unclear, please explain.
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Reviewer comment for Spectrum01578-22 (Caffeic Acid Phenethyl Ester (CAPE) Inhibits Cross-Kingdom Biofilm Formation of Streptococcus mutants and Candida Albicans)
It is well known that Candida albicans and Streptococcus mutans interact to form robust biofilm, which in turn degrade enamel to cause cavities. Therefore, determining mechanisms to disrupt and these biofilms is crucial in regulating their disease outcome. The current manuscript employed Caffeic Acid Phenethyl Ester (CAPE), a naturally existing compound, to disrupt and kill both bacteria and their cross-kingdome biofilm formation. Authors carefully monitored planktonic and biofilm growth and EPS formation in presence of increasing concentration of CAPE. Although some of the explanation and argument of the data can be improved (see below), this article can help progressing the current field. Comments 1. Authors argue that CAPE suppressed growth, biofilm formation and EPS synthesis via interactions with the two species. However, in their experiments, they did not show direct interaction of the CAPE with the two species. This has to be shows by actual experiment. 2. How using CAPE against biofilm formation will affect its other activities in eukaryotic organisms? Most importantly, CAPE has immunomodulatory activity, which may impact the eukaryotic host physiology unintended way. In addition to testing cytotoxicity, how immune activity may go a long way for using this product as therapeutic. 3. CAPE has effect on cross-Kingdome biofilm formation of S.mut and C. albicans in vitro experiments. How about in vivo biofilms? How this cross-kingdome biofilm is different in in vivo and whether CAPE can still function in the native environment? 4. In Figure 1, panel A and B show that C. albicans grow in 40 ug/mL CAPE despite in low numbers. However, in Figure 1D, OD600 show that under the same concentration, no growth can be observed. What is the reason? 5. In Figure 4, can the decrease in EPS formation be due to not CAPE inhibiting EPS formation but CAPE killing the bacteria? Both biomass and EPS amount decreased simultaneously. In other words, can decrease in EPS be due to lower number of bacteria? How can the authors argue one versus the other? The given experiment is not enough to distinguish between these two possibilities. Perhaps, the authors need to normalize EPS amount to per bacterial cell bases to argue this point? 6. What is the CAPE biofilm penetration depth? Can you label CAPE and measure how deep it can penetrate the biofilm? 7. Authors argue that HOK cell viability was minimally affected by CAPE, but Figure 6 shows statistically significant difference in all concentrations tested when compared to control (no CAPE experiment). This is counterintuitive and show that CAPE are toxic to eukaryotic cells. Also, at 40 and 80 ug/mL (MIC), the HOK cells had 80% viability which means 20% of the HOK cells were killed. By therapeutic standards, this is very high and cannot be used in humans. 8. What is the MIC of CAPE for other strains of S.mut and C.albicans. Are they similar to UA159 and SC5314? How did you pick these strains for the study? 9. How can a CFU be determined from a biofilm in Figure 2B? Don't they clump-up and form aggregates, giving false CFU? 10. In Figure 5 and lines 182-188, authors need to explain what gtf genes do? And why they decide to test for the expression level of these genes? Did the authors do comprehensive screen for other cariogenic genes? 11. It will go a long way to show additional oral epithelial cell tests. HOK cells are one of many oral keratinocytes and they may show different effect compare to other cell lines.

Response to Reviewer #2
Dear Reviewer, We thank you for your professional review of our article. There were several issues that needed to be addressed. According to your helpful suggestions, we have made extensive modifications to our previous manuscript and supplemented additional data to make our results more convincing; the detailed corrections are listed. Please see below, in blue text, our point-by-point responses to your comments and concerns. All page numbers refer to the revised manuscript file with tracked changes.

Comment 1:
Authors argue that CAPE suppressed growth, biofilm formation and EPS synthesis via interactions with the two species. However, in their experiments, they did not show direct interaction of the CAPE with the two species. This has to be shows by actual experiment.

Response 1:
We gratefully appreciate your careful review and noting our inaccurate statement. We apologize for the misunderstanding caused by our inaccurate representation of the role of CAPE and would like to explain it in more detail.
According to reported studies (1-3), the interactions of S. mutans and C. albicans can enhance cariogenicity, which is consistent with our research basis. Our study displayed the inhibitory effects of CAPE on S. mutans and C. albicans, which corresponded to previous studies (4,5). Therefore, we hypothesized that CAPE could also play an inhibitory role in the co-culture systems. Through confirmed experiments, CAPE can indeed reduce the cariogenicity of S. mutans and C. albicans co-culture systems by suppressing growth, biofilm formation and EPS synthesis.
However, our results were misinterpreted as the interaction of CAPE with the two species. We apologize for the inaccurate text and have made the following two modifications.
The first modification is shown in Lines 31-33.
We changed "As a result, CAPE suppressed the growth, biofilm formation and EPS synthesis of C.
albicans and S. mutans via interactions with the two species." to "The results showed that CAPE suppressed the growth, biofilm formation and extracellular polysaccharides (EPS) synthesis of C. albicans and S. mutans in the co-culture system of the two species." The second modification is shown in Lines 89-91.
We changed "In general, the number of studies on the interaction of CAPE with C. albicans and S.
mutans and the effects of CAPE on the cariogenic virulence of cross-kingdom biofilms is limited, which prompted us to study this topic further." to "In general, the effects of CAPE on the cariogenic virulence of cross-kingdom biofilms formed by C. albicans and S. mutans are still unclear, which prompted us to study this topic further."

Comment 2:
How using CAPE against biofilm formation will affect its other activities in eukaryotic organisms? Most importantly, CAPE has immunomodulatory activity, which may impact the eukaryotic host physiology unintended way. In addition to testing cytotoxicity, how immune activity may go a long way for using this product as therapeutic.

Response 2:
Thank you for your valuable suggestion. As a type of multifunctional active substance, CAPE has been extensively studied (6,7), especially with regard to its immunomodulatory activity, as you mentioned. Our research concentrated on the antimicrobial activity of CAPE and its potential application value in the prevention of severe caries, which was consistent with most previous studies on the antibacterial and antifungal effects of CAPE (4,(8)(9)(10).
On the basis of the current relevant report (11,12), antimicrobial agents are usually used as mouthwash additives or toothpaste additives in daily oral protection. Taking the commonly used hydrogen peroxide (H 2 O 2 ) mouthwash as an example (13), although H 2 O 2 has important biological functions as a reactive oxygen species, it is still used as a mouthwash additive.
Therefore, from the perspective of this study, we will consider the further application of relevant research in the future from the dosages, dosage form, dosage regimen and other aspects of CAPE to conduct a more comprehensive evaluation. Accordingly, for further study on the immunomodulatory activity of CAPE, we provide a supplementary exposition in the Discussion of the article (Line 308-311).

Comment 3:
CAPE has effect on cross-Kingdome biofilm formation of S. mutans and C. albicans in vitro experiments. How about in vivo biofilms? How this cross-kingdom biofilm is different in in vivo and whether CAPE can still function in the native environment?

Response 3:
We sincerely thank you for this important comment, which we also considered at the beginning of our research design. Therefore, we will explain our consideration in detail as follows.
Although CAPE has significant advantages in biological activities, its low water solubility leads to poor bioavailability, which limits its therapeutic application and in vivo model construction (6,14). Studies have shown that CAPE can be hydrolysed into caffeic acid by esterase in vivo (15), which observably limits the effectiveness and often requires high doses to achieve the therapeutic effect.
In our research on the antimicrobial activity of CAPE, although we agree that the in vivo biofilm model is an important consideration, it is beyond the scope of this study.
The oral cavity is a moist and dynamic microenvironment, which presents in vivo experiments with the potential inefficiency of the simple CAPE dosage form.
Currently, applications of biomaterials and modern drug delivery systems are gradually being developed to address CAPE's poor solubility and bioavailability. For example, the application of nanocarriers for CAPE could achieve better therapeutic effects at lower doses of CAPE (16). This explanation of the limitations of CAPE and the prospects for future research are mentioned in the Discussion section (Lines 303-311). Furthermore, we are planning to explore the combination of CAPE and nanotechnology in the microenvironment.

Comment 4:
In Figure 1, panel A and B show that C. albicans grow in 40 ug/mL CAPE despite in low numbers. However, in Figure 1D, OD600 show that under the same concentration, no growth can be observed. What is the reason?

Response 4:
Thank you for this question, which I will address as follows.
First, we carefully checked the data in Figure 1 and ensured its authenticity. The plate coating experiment shown in panels A and B is based on the determination of MIC, which is referred to as a more intuitive and accurate supplement to the antimicrobial effect of CAPE at gradient concentrations. Therefore, the plate-coating results tend to reflect the more subtle microbial growth in the system.
The growth of C. albicans at 40 μg/mL CAPE shown in panel D presented as a flat curve, similar to the stationary phase in which the bacterial growth reached a plateau as the number of dying cells equalled the number of dividing cells. This finding illustrated that the growth and proliferation of C. albicans were initially significantly inhibited by CAPE; thus, the fungal density remained almost constant. However, the sluggish growth of C. albicans does not mean that the C. albicans in the original system were completely killed by CAPE; there is no obvious recession that would be considered the death phase. Therefore, the CAPE-treated group at 40 μg/mL showed the presence of C. albicans in panels A and B.
In view of our unclear text in the Results, we have modified the text as shown in Lines 115-117.
We changed "With regard to the growth of C. albicans, the optical density in the system indicated that C. albicans hardly grew with 1/2 MIC CAPE." to "With regard to the growth of C. albicans, the optical density in the system indicated that C. albicans hardly proliferated and grew sluggishly with 1/2 MIC CAPE."

Comment 5:
In Figure 4, can the decrease in EPS formation be due to not CAPE inhibiting EPS formation but CAPE killing the bacteria? Both biomass and EPS amount decreased simultaneously. In other words, can decrease in EPS be due to lower number of bacteria? How can the authors argue one versus the other? The given experiment is not enough to distinguish between these two possibilities. Perhaps, the authors need to normalize EPS amount to per bacterial cell bases to argue this point?

Response 5:
We appreciate this meaningful question, and we will elaborate on these two valid arguments.
On the one hand, we have directly confirmed that CAPE can kill bacteria in biofilms through the live/dead bacterial viability assay using CLSM. On the other hand, the qRT-PCR assay was used to verify the inhibitory effect of CAPE on EPS synthesis, rather than decreasing the amount of EPS simply by reducing the living bacteria. The results demonstrated that CAPE could significantly downregulate the expression level of Gtf genes (gtfB, gtfC, gtfD), which guide the production of EPS through the synthesis of Gtf enzymes. In other words, the outcomes of the qRT-PCR assay indirectly proved that CAPE could inhibit EPS formation by controlling the expression of corresponding cariogenic genes.
Consequently, we believe that CAPE reduces biofilm biomass by simultaneously killing bacteria and inhibiting EPS formation. These methods have also been demonstrated in other studies on the antimicrobial properties of natural products or extracts (17,18).
Thank you again, and we sincerely hope our response has resolved this question.

Comment 6:
What is the CAPE biofilm penetration depth? Can you label CAPE and measure how deep it can penetrate the biofilm?

Response 6:
We appreciate your valuable comment.
In our opinion, CAPE biofilm penetration depth is the key to the effectiveness of CAPE because CAPE penetration is directly related to microbial inactivation and the control of biofilms (19). In our research, confocal laser scanning microscopy (CLSM) was applied to observe the stained biofilms, as shown in Figure 3. This procedure only illustrated the changes in the surface structure of biofilms and the quantitative analysis of fluorescence.
Therefore, according to your suggestion, we have made some supplements as follows.
Briefly, we used ImageJ COMSTAT software to reconstruct the three-dimensional architecture of biofilms and quantitatively measure the thickness of biofilms to evaluate CAPE biofilm penetration depth, which is consistent with the reported references (9,20).  to "Three points were randomly selected for the inspection of every specimen, the quantified statistical results of fluorescence intensity and the biofilm thickness of the three-dimensional reconstruction images were assessed using ImageJ COMSTAT software." We also added these important references (9,20).
In the Results section, we added the corresponding results, as shown in Lines 162-170 as follows.
"The thickness of S. mutans and cross-kingdom biofilms affected by CAPE is displayed in Fig. 4. The three-dimensional reconstruction of the biofilm architecture indicated that the thickness reduced with the increase in CAPE concentration. The biofilms generated in the control group were approximately 20 μm on glass coverslips without antimicrobial treatment. With the penetration of CAPE, the biofilm integrity was destroyed, so that both S. mutans and cross-kingdom biofilms significantly became thinner. Furthermore, as S. mutans and C. albicans were inactived and eliminated, the penetration depth of CAPE into biofilms deepened. Generally speaking, the effectiveness of CAPE is closely related to the inhibition of microbial growth and the penetration of biofilms."

Comment 7:
Authors argue that HOK cell viability was minimally affected by CAPE, but Figure 6 shows statistically significant difference in all concentrations tested when compared to control (no CAPE experiment). This is counterintuitive and show that CAPE are toxic to eukaryotic cells. Also, at 40 and 80 ug/mL (MIC), the HOK cells had 80% viability which means 20% of the HOK cells were killed. By therapeutic standards, this is very high and cannot be used in humans.

Response 7:
Thank you for your valuable suggestion; our explanation is as follows.
In this study, we chose chlorhexidine (CHX) as the positive control. CHX is considered the gold standard for application in oral rinses and has been extensively used to remove plaque biofilms in clinical dentistry; in particular, a 0.12% concentration of CHX is used to prevent caries occurrence and progression (4,21,22).
However, its cytotoxicity remains a disadvantage that has not been well resolved (23).
According to reported research (24), a low concentration of CHX (0.05%) resulted in less than 40% oral keratinocyte viability.
We carefully verified the data in Figure 6 to ensure its authenticity. At a 20 μg/mL concentration of CAPE, human oral keratinocytes (HOKs) exhibited approximately 90% viability. Additionally, the HOK cells had more than 80% viability at 40 μg/mL and 80 μg/mL (MIC). According to the classification of cytotoxicity (25), extracts are considered severely, moderately or slightly cytotoxic when the viability relative to the controls is less than 30%, between 30% and 60%, or greater than 60%, respectively.
Therefore, CAPE illustrated "slight cytotoxicity" in our research instead of good biocompatibility, which was corrected as shown in Line 292.
Although all CAPE-treated groups showed a significant difference from the control group, the 80% oral keratinocyte viability with CAPE treatment was significantly higher than that treated with a low concentration of CHX below 40%. Additionally, our supplementary susceptibility assay data show that the MIC of CHX ranged from 4×10 3 to 8×10 3 mg/mL for S. mutans and C. albicans, which is significantly higher than the MIC concentration of CAPE. Therefore, our research suggested that the biocompatibility of CAPE is acceptable and has application potential.
Accordingly, we have made modifications in the Abstract, Materials and Methods, Results, and Discussion.
In the Abstract, we changed "good" to "acceptable" in Lines 34-35.
In the Materials and Methods, as shown in Lines 342-344, In the Results section, there are three changes.
The first addition is shown in Lines 103-105.
We added "The MIC of CHX ranged from 4×10 3 to 8×10 3 mg/mL for both strains as the positive control, which was significantly higher than the MIC concentration of The second modification is shown in Line 203.
We changed the title "CAPE showed good biocompatibility" to "The biocompatibility of CAPE".
The third modification is shown in Lines 208-211.
We changed "The viability of HOK cells was above 80% with 40 μg/mL and 80 μg/mL CAPE and above 90% on average with a CAPE concentration of 20 μg/mL, which indicated the good biocompatibility of CAPE." to "The viability of HOK cells was above 80% with 40 μg/mL and 80 μg/mL CAPE and above 90% on average with a CAPE concentration of 20 μg/mL. Compared with CHX, which exhibited obvious cytotoxicity (see the Discussion section), the biocompatibility of CAPE is acceptable." In the Discussion, We added "The biocompatibility of CAPE has been reported by previous studies (26).
In our research, CAPE showed slight cytotoxicity to human oral keratinocytes (HOKs), according to the classification (25), which rates extracts as having severe, moderate or slight cytotoxicity when the viability relative to the control groups is less than 30%, between 30% and 60%, or greater than 60%, respectively. It is worth noting that CHX is currently considered one of the most common antiseptics in dentistry and the gold standard agent for the application of oral rinses; in particular, the clinical concentration of 0.12% CHX is used to remove plaque biofilm (21,22,27). However, its cytotoxicity has been widely reported, especially the obvious reduction of CHX against HOKs and fibroblast cell numbers in a short time (28). Barbara Azzimonti et al. (24) also emphasized that a low concentration of CHX (0.05%) resulted in less than 40% mucosal keratinocytes viability. Thus, our research suggested that the biocompatibility of CAPE is acceptable and has application potential." in Lines

Comment 8:
What is the MIC of CAPE for other strains of S. mutans and C. albicans. Are they similar to UA159 and SC5314? How did you pick these strains for the study?

Response 8:
Thank you very much for this question. The selection of strains was taken into account in the experimental design stage. We explain the process in detail as follows.
First, both the S. mutans strain UA159 (ATCC 700610) and the C. albicans strain SC5314 (ATCC 10691) have been extensively applied as universal reference strains from American Type Culture Collection (ATCC). The reference strains have comprehensive and typical microbiological characteristics, which can be used as the standard of quality control in microbiological examination and scientific research.
The genome of S. mutans strain UA159 has been sequenced (29). Based on the UA159 genome, studies with comparative genomic hybridization (CGH) have shown a high degree of content variation among strains, with some isolates lacking up to 20% of the genes present in the reference strain UA159 (30,31). Thus, to make our research more comprehensive, standardized and valuable in application, the S. mutans strain UA159 was selected, which is consistent with other studies (17,32,33).
Additionally, the C. albicans strain SC5314 was used in this study as a reference strain and is known to be severely invasive with strong biofilm formation (34)(35)(36).
This use is also in accordance with other reports (35)(36)(37)(38), especially studies of the effects against biofilms.
Therefore, in our opinion, this study on the effects of CAPE on the S. mutans strain UA159 and the C. albicans strain SC5314 would be meaningful for clinical applications. Further effects of CAPE for other strains of S. mutans and C. albicans will be investigated in subsequent studies.

Comment 9:
How can a CFU be determined from a biofilm in Figure 2B? Don't they clump-up and form aggregates, giving false CFU?

Response 9:
Thank you very much for noting that we missed critical experimental steps in the Materials and Methods. We will explain this process clearly as follows.
In the CFU-counting assay, the biofilm aggregates were scraped off from the bottom of the microwells. According to reported protocols (39,40), the samples were then mixed thoroughly by vortexing with sterile PBS so that the suspension could be used to dilute, incubate and count colonies.
Accordingly, we having provided this missing information and added references, as shown in Lines 393-398.
We changed "The adherent biofilms were then removed from the floor of each microwell, resuspended in sterile PBS and diluted 10 6 -fold to count S. mutans colonies and 10 4 -fold to count C. albicans colonies." to "The adherent biofilms were then removed from the floor of each microwell and thoroughly mixed by vortexing with 200 μL of sterile PBS. After resuspension, each sample was diluted 10 6 -fold to count S. mutans colonies and 10 4 -fold to count C. albicans colonies."

Comment 10:
In Figure 5 and lines 182-188, authors need to explain what gtf genes do? And why they decide to test for the expression level of these genes? Did the authors do comprehensive screen for other cariogenic genes?

Response 10:
Thank you for this valuable suggestion. We will explain this aspect clearly.
Glucosyltransferases (Gtfs) play crucial roles in the occurrence and development of S. mutans-mediated caries, including early childhood caries (41). Generally, Gtfs can produce extracellular polysaccharides (EPS) to promote biofilm formation and mediate the adhesion of cariogenic bacteria (4,42). The gtf genes (gtfB, gtfC, gtfD) encode three Gtf enzymes (GtfB, GtfC, GtfD) produced by S. mutans. The expression of the three gtf genes is distinct but related according to a reported study (41). Briefly, the expression level of gtf genes is closely associated with EPS synthesis.
Furthermore, at the beginning of the study design, we extensively screened cariogenic genes to determine the expression level of these three gtf genes, which are closely related to the cariogenicity of S. mutans. This finding is consistent with other studies on cariogenic genes (18,43).
According to your suggestion, we have added the following text, as shown in Lines

193-195.
"Gtfs, as enzymes that ferment sugar, catalyzing the transformation of glucosyl groups, contribute to the synthesis of EPS by S. mutans. The expression level of gtf genes is closely related to EPS synthesis." Additionally, we revised the text in Lines 199-201 as follows.

Comment 11:
It will go a long way to show additional oral epithelial cell tests. HOK cells are one of many oral keratinocytes and they may show different effect compare to other cell lines.

Response 11:
We appreciate your valuable comment; we will explain our consideration as follows.
Oral epithelial cells can be divided into keratinocytes and nonkeratinocytes according to whether they participate in keratinization. Oral keratinocytes, which were used in our study, act as the major barrier to oral diseases, protecting local cells from physical, microbial, and chemical damage. Furthermore, oral keratinocytes can potentially participate in controlling oral infections through the inflammatory process according to the report (44). Consequently, in our opinion, the application of oral keratinocytes for cell viability research can be representative, which is consistent with other studies on the effects of natural products or extracts against oral microbes (22,45,46).
Subsequently, based on the measurement of cell viability as the method of cytotoxicity evaluation, we will conduct a more comprehensive assessment of CAPE's effects on inflammatory reactions in animal or clinical models. This aspect deserves to be investigated for more comprehensive understanding of biocompatibility in our subsequent studies, which is consistent with previous reports (47,48).
We would like to take this opportunity to thank you for all your time involved and this opportunity for us to improve the manuscript. We hope you will find this revised

Response to Reviewer #3
Dear Reviewer, We sincerely thank you for your professional review work and your valuable feedback, which we have used to improve the quality of our manuscript. According to your kind suggestions, we have made extensive modifications to our manuscript, and the detailed corrections are listed. Please see below, in blue text, our point-by-point responses to your comments and concerns. All page numbers refer to the revised manuscript file with tracked changes.

Comment 1:
Abstract: ASM Journals not use sectioned abstracts, please combine and reword accordingly.

Response 1:
Thank you for the suggestion. We have revised the Abstract as follows (Lines 19-37).

Comment 2:
Importance: "closely-related to the cross-kingdom", this sentence has awkward phrasing as currently written, please revise.

Response 2:
Thank you for your comment. We have modified this sentence to make the meaning more accurate and clearer. The revised text reads as follows (Lines 39-40).
We changed "Severe dental caries is closely related to the cross-kingdom biofilm formed by S. mutans and C. albicans." to "Severe dental caries is a multimicrobial infectious disease that is strongly induced by the cross-kingdom biofilm formed by S. mutans and C. albicans."

Comment 3:
Line 57: please change "the key pathogen" to "a key pathogen", as many researchers in the field would consider this an exaggeration of the role of S. mutans, as cases of caries without detectable levels of S. mutans are routine.

Response 3:
Thank you for your careful reading and kind reminder. We completely agree that it is inaccurate to regard S. mutans as the key pathogen of dental caries. Thus, we have made the modification according to your opinion, as shown in Line 55.

Comment 4:
Line 110: "corresponding colony variations" please clarify or change? Do you mean number of CFUs or differences in the actual colony morphology?

Response 4:
Thank you for noting this issue. We were attempting to emphasize the changes in the number of colonies, as shown by the CFU assay.
Therefore, we have corrected this inaccurate text as follows (Line 110).
We changed "corresponding colony variations" to "corresponding variations in colony numbers".

Comment 5:
Line 156: "was affected by CAPE" awkward phrasing again here...I think you mean something more like "a reduction in biofilm accumulation correlated with increasing concentration of CAPE"

Response 5:
We gratefully appreciate your careful review and noting our inaccurate statement. We apologize for the misunderstanding caused by awkward phrasing.
According to your suggestion, we made the modification shown in Lines 156-157.
We changed "the observable reduction in biofilm accumulation was affected by CAPE." to "the observable reduction in biofilm accumulation correlated with increasing CAPE concentrations."

Comment 6:
qRT-PCR: This is important! If I understand the methods correctly, the relative expression of gtfB, C, and D in each sample is "relative" to the corresponding 0µg CAPE sample. Since there are clearly fewer cells, and more dead cells in the higher CAPE concentration samples, you would expect less gtf RNA, and indeed less RNA overall in those samples. Crucially, using this method, you therefore cannot hypothesize that CAPE specifically reduced the transcription of gtf genes. You would need to control for the differences in cell viability and/or also compare to the RNA levels of other genes. Please either add experiments with the proper controls or remove this experiment from the study.

Response 6:
Thank you for noting this issue; we will explain this method.
In our research, S. mutans UA159 16S rRNA was used as an internal control, according to previous studies (1)(2)(3). It has been reported that the bacterial 16S rRNA gene, as the reference gene, can be applied to calculate the relative abundance of the specific 16S rRNA genes of the target bacteria and the relative expression level of the functional genes (4). The relative expression level of functional genes was simply defined as the ratio of DNA fragments detected to the total bacteria detected via qPCR.
Accordingly, our research utilized S. mutans UA159 16S rRNA as the reference to calculate the relative expression level of the gtf genes (gtfB, gtfC, gtfD), both in the control group without CAPE treatment and in CAPE-treated groups with different concentrations. Through the measurement of real-time qPCR, the results could be interpreted as the average amount of target gene expression per bacterium, which was defined as the relative mRNA expression levels of gtf genes.
We apologize for the misunderstanding caused by the inaccurate statement. Therefore, we made the modification shown in Lines 463-464.
We changed "The expression levels of gtfB, gtfC and gtfD mRNA were determined by the qRT-PCR assay, with S. mutans UA159 used as the reference." to "The relative expression levels of gtfB, gtfC and gtfD mRNA were determined using the qRT-PCR assay, with S. mutans UA159 16S rRNA used as the reference."

Comment 7:
Biocompatibility: It is not clear from the current manuscript what current "acceptable" levels of CCK-8 viability are, and how much toxicity in the assay might translate to toxicity in vivo. Please either remove claims of "good biocompatibility" or add references explaining what "good biocompatibility" is and how the results here compare to it.

Response 7:
Thank you very much for noting this issue.
According to your valuable suggestions, we chose chlorhexidine (CHX) as the positive control in our research. CHX is considered the gold standard substance for application in oral rinses and has been extensively used to remove plaque biofilms in clinical dentistry; in particular, a 0.12% concentration of CHX is used to prevent the occurrence and progression of caries (5-7). However, its cytotoxicity remains a disadvantage that has not been well resolved (8). According to reported research (9), a low concentration of CHX (0.05%) resulted in oral keratinocyte viability of less than 40%.
In our research, at a 20 μg/mL concentration of CAPE, human oral keratinocytes (HOKs) exhibited approximately 90% viability. Additionally, the HOK cells had more than 80% viability at 40 μg/mL and 80 μg/mL (MIC). According to the classification of cytotoxicity (10), extracts are considered severely, moderately or slightly cytotoxic when the viability relative to the controls is less than 30%, between 30% and 60%, or greater than 60%, respectively. Therefore, CAPE illustrated "slight cytotoxicity" in our research instead of good biocompatibility, which has been corrected as shown in

Line 292.
Although all CAPE-treated groups showed a significant difference from the control group, the 80% oral keratinocyte viability with CAPE treatment was significantly higher than that of cells treated with a low concentration of CHX (below 40% viability). Additionally, our supplementary susceptibility assay data show that the MIC of CHX ranged from 4×10 3 to 8×10 3 mg/mL for S. mutans and C. albicans, which was significantly higher than the MIC concentration of CAPE. Therefore, our research suggested that the biocompatibility of CAPE is acceptable and has application potential.
Accordingly, we have made the supplements and modifications in the Abstract, Materials and Methods, Results, and Discussion.
In the Abstract, we changed "good" to "acceptable" in Lines 34-35.
In Materials and Methods, as shown in Lines 342-344.
We changed "CHX (Sigma-Aldrich, Steinheim, Germany) replaced CAPE in microcells as the positive control according to reported protocols" to "As the positive control, CHX (Sigma-Aldrich, Steinheim, Germany) replaced CAPE in microcells at concentrations ranging from 1.0×10 3 to 128.0×10 3 mg/mL according to reported protocols." In the Results section, there are three changes.
The first addition is in Lines 103-105.
We added "The MIC of CHX ranged from 4×10 3 to 8×10 3 mg/mL for both strains as the positive control, which was significantly higher than the MIC concentration of The second modification is shown in Line 203.
We changed the title "CAPE showed good biocompatibility" to "The biocompatibility of CAPE".
The third modification is in Lines 208-211.
We changed "The viability of HOK cells was above 80% with 40 μg/mL and 80 μg/mL CAPE and above 90% on average with a CAPE concentration of 20 μg/mL, which indicated the good biocompatibility of CAPE." to "The viability of HOK cells was above 80% with 40 μg/mL and 80 μg/mL CAPE and above 90% on average with a CAPE concentration of 20 μg/mL. Compared with CHX, which exhibited obvious cytotoxicity (see the Discussion section), the biocompatibility of CAPE is acceptable." In the Discussion, We added "The biocompatibility of CAPE has been reported by previous studies (11).
In our research, CAPE showed slight cytotoxicity to human oral keratinocytes (HOKs), according to the classification (10), which rates extracts as having severe, moderate or slight cytotoxicity when the viability relative to the control groups is less than 30%, between 30% and 60%, or greater than 60%, respectively. It is worth noting that CHX is currently considered one of the most common antiseptics in dentistry and the gold standard agent for the application of oral rinses, in particular, the clinical concentration of 0.12% CHX is used to remove plaque biofilm (6,7,12). However, its cytotoxicity has been widely reported, especially the obvious reduction of CHX against HOKs and fibroblast cell numbers in a short time (13). Barbara Azzimonti et al. (9) also emphasized that a low concentration of CHX (0.05%) resulted in less than 40% mucosal keratinocytes viability. Thus, our research suggested that the biocompatibility of CAPE is acceptable and has application potential." in Lines

Comment 8:
Line 218: please remove "promising", I think that's a bit overboard at this stage.

Response 8:
Thank you for the detailed review, and we agree with your suggestion.
Therefore, we changed "this promising natural product" to "this natural product", as shown in Lines 231-232.

Response 9:
Thank you again for noting the discussion about the expression of cariogenic gene by qRT-PCR. We hope that response #6 above can resolve these important and valuable questions.
We have also modified the inaccurate text, as shown in Lines 265-271.

Response 10:
We apologize for our careless error; thank you so much for your comment.
We have corrected "implants" to "plants", as shown in Line 279.

Response 11:
Thank you again for noting our inaccurate description of the biocompatibility of CAPE. According to your suggestions, as shown in comment #6 above, we have added "The biocompatibility of CAPE has been reported by previous studies (11). In our research, CAPE showed slight cytotoxicity to human oral keratinocytes (HOKs), according to the classification (10), which rates extracts as having severe, moderate or slight cytotoxicity when the viability relative to the control groups is less than 30%, between 30% and 60%, or greater than 60%, respectively. It is worth noting that CHX is currently considered one of the most common antiseptics in dentistry and the gold standard agent for the application of oral rinses; in particular, the clinical concentration of 0.12% CHX is used to remove plaque biofilm (6,7,12). However, its cytotoxicity has been widely reported, especially the obvious reduction of CHX against HOKs and fibroblast cell numbers in a short time (13). Barbara Azzimonti et al. (9) also emphasized that a low concentration of CHX (0.05%) resulted in less than 40% mucosal keratinocytes viability. Thus, our research suggested that the biocompatibility of CAPE is acceptable and has application potential." in Lines 291-302.

Comment 12:
Lines 371-372: Since I don't believe that YPD and BHI are selective, how do you differentiate between colonies of S. mutans and C. albicans by plating on solid media from the co-culture? This is unclear, please explain.

Response 12:
We sincerely thank you for this important comment, which we also considered at the beginning of our research design.
In the CFU-counting assay for planktonic S. mutans and C. albicans, the cocultured suspensions were diluted 10 4 -fold to count C. albicans colonies, while S. mutans could not be recognized as having a colony morphology. Conversely, S. mutans colonies were counted with 10 6 -fold dilution of the cocultured suspensions, while the larger C. albicans cells rarely existed in the diluent. According to a reported study (3), this method for CFU counting was applied in our research, as shown in Lines 357-362. Therefore, we added the corresponding reference.
Furthermore, in the CFU-counting assay for quantification of biofilm biomass, the biofilm aggregates were scraped off from the bottom of the microwells. In accordance with reported protocols (3,14), the aggregates were then mixed thoroughly by vortexing with sterile PBS so that the suspension could be used to dilute, incubate and count colonies with the same method. As shown in Lines 393-398, we have provided more detailed text and added these references.
We changed "The adherent biofilms were then removed from the floor of each microwell, resuspended in sterile PBS and diluted 10 6 -fold to count S. mutans colonies and 10 4 -fold to count C. albicans colonies." to "The adherent biofilms were then removed from the floor of each microwell and thoroughly mixed by vortexing with 200 μL of sterile PBS. After resuspension, each sample was diluted 10 6 -fold to count S. mutans colonies and 10 4 -fold to count C. albicans colonies."